The main objective of this work is to elucidate the biochemical mechanisms regulating the GTP-binding protein ARF and, in so doing, to further our understanding of the role of ARF in membrane traffic. ARF's interaction with target proteins is dependent on phospholipids. A phosphatidylinositol 4,5-bisphosphate (PIP2) binding site on Arf has been tentatively identified as comprising residues 178 and 181 of the carboxy terminal domain. Binding of PIP2 to this site promotes interaction with Arf GAP. This is consistent with the hypothesis that specific lipids may direct Arf interactions with specific proteins and thereby provide a means of ordering Arf-dependent events in membrane traffic. Having found that PIP2 and PA were required for ARF - ARF GAP interaction, we have been able to make some progress in the purification of the latter protein. The protein has now been purified approximately 2,000 fold and has been shown to be distinct from coat proteins and the other known Arf GAP. The two Arf GAPs had similar substrate preferences but differed in their regulation by phospholipids. Multiple GAPs may independently regulate Arf at Arf's multiple sites of action.